ADHESIVE REGULATION DURING CELL MIGRATION

整合素和其他细胞表面粘附受体通过介导细胞外基质（ECM）和肌动蛋白细胞骨架之间的相互作用来调节细胞的迁移和侵袭。在迁移细胞中，整合素与细胞骨架的相互作用在时间和空间上都受到高度调节。我们研究的长期目标是鉴定和表征在迁移过程中调节整合素-细胞骨架相互作用的分子。我们之前的研究发现钙依赖性蛋白酶calpain是这些相互作用的调节因子。抑制钙蛋白酶活性可抑制细胞迁移和侵袭。calpain与其他细胞通路结合调节细胞迁移的方法在很大程度上仍然未知，将在具体目的I和II中进行研究。为了鉴定调节细胞迁移的其他蛋白质，我们采用了表达克隆方案。初步研究已经确定了候选调节因子，包括与蛋白激酶c结合蛋白RACK1序列同源的蛋白。为了表征迁移过程中调节整合素-细胞骨架相互作用的分子机制，我们提出以下具体目标：1 .表征迁移细胞中调节mu-calpain的时空分布的机制。在迁移过程中，调节mu-calpain细胞内分布的粘附和信号通路将在活细胞中使用mu-calpain- gfp融合蛋白进行研究。2。描述钙蛋白酶功能与整合素-细胞骨架相互作用之间的关系。利用整合素突变体与细胞骨架蛋白（talin或filamin）结合，研究钙蛋白酶活性改变对整合素介导的迁移和信号转导的影响。3。鉴定和表征在细胞迁移过程中调节整合素-细胞骨架相互作用的新蛋白。表达克隆将用于鉴定在迁移过程中调节整合素-细胞骨架相互作用的其他蛋白质。在早期筛选中发现的RACK1同源物将被表征。这些研究将增强我们对调节细胞迁移的分子机制的理解，并提供与肿瘤侵袭和转移等病理过程相关的信息。

Integrins and other cell surface adhesion receptors regulate cell migration and invasion by mediating interactions between the extracellular matrix (ECM) and the actin cytoskeleton. Integrin- cytoskeleton interactions are highly regulated both temporally and spatially in migrating cells. The long term goal of our research is to identify and characterize the molecules that regulate integrin-cytoskeleton interactions during migration. Our previous studies identified the calcium-dependent protease calpain as a regulator of these interactions. Inhibition of calpain activity suppresses cell migration and invasiveness. The means by which calpain integrates with other cellular pathways to regulate cell migration remain largely unknown, and will be investigated in specific aims I and II. To identify other proteins that modulate cell migration we have employed an expression cloning scheme. Preliminary studies have identified candidate regulators, including a protein with sequence homology to the protein kinase C-binding protein RACK1. To characterize the molecular mechanisms that regulate integrin- cytoskeletal interactions during migration we propose the following Specific aims: I. Characterize the mechanisms that regulate the temporal and spatial distribution of mu-calpain in migrating cells. The adhesive and signaling pathways that modulate the intracellular distribution of mu-calpain during migration will be studied in live cells using a mu-calpain-GFP fusion protein. II. Characterize the relationship between mu- calpain function and integrin-cytoskeletal interactions. Using integrin mutants with well characterized binding to the cytoskeletal proteins talin or filamin, the effects of altered calpain activity on integrin-mediated migration and signal transduction will be examined. III. Identify and characterize novel proteins that regulate integrin-cytoskeletal interactions during cell migration. Expression cloning will be used to identify additional proteins that regulate integrin-cytoskeletal interactions during migration. The RACK1 homolog identified in early rounds of screening will be characterized. These studies will enhance our understanding of the molecular mechanisms that regulate cell migration and provide information relevant to pathological processes such as tumor invasion and metastasis.